Hepatitis delta virus (HDV) is a significant human pathogen with 20 million chronic carriers worldwide. HDV is a natural subviral agent of human hepatitis B virus (HBV). HDV uses HBV envelope proteins to form virions and infect hepatocytes. In naturally infected liver, HDV co-exists with HBV. Worldwide, chronic HBV infection is a main risk factor of hepatocellular carcinoma (HCC) and is associated with >50% of all HCC cases. Concomitant HDV infection is able to inflict additional liver damage, often resulting in accelerated liver disease and more fast/frequent cirrhosis. Chronic HBV/HDV carriers have a three-fold increased risk of HCC incidence, and develop HCC about 14 years earlier as compared to the carriers of HBV only. Currently, no drugs directly targeting HDV are in use, and a number of anti-HBV drugs do not block HDV infection. HBV-infected individuals support HDV infection regardless of the presence of HBV replication markers. In livers chronically infected with HBV, up to 90% of hepatocytes can appear free of HBV replication markers, but a significant number of hepatocytes and most HCCs contain integrated HBV DNA. The envelope proteins can be produced from the integrated HBV DNA even when HBV replication is ceased. Since HDV takes only the envelope proteins from HBV, we anticipate that HDV can persist in absence of HBV replication, if the envelope proteins are available. Since alterations (rearrangements, mutations, deletions, etc.) are frequently observed in integrated HBV DNAs and in HBV integrant-derived mRNAs for the envelope proteins (ine mRNAs), it remains to be determined whether the envelope proteins translated from the ine mRNAs can support HDV assembly and infectivity. Therefore, this application will test the hypothesis that persistent HDV infection can be maintained exclusively via production of functional envelope proteins from integrated HBV DNA in the absence of HBV replication. Since a double-stranded linear HBV DNA genome is a main substrate for integration, HBV promoters and coding sequences for the envelope proteins may remain intact in the integrant, while HBV poly(A) signal/site are likely not present downstream. However, HBV envelope proteins can be expressed from integrants, when polyadenylation is facilitated by adjacent downstream host sequences. We propose to clone from HBV-infected liver tissues and matching HBV-induced HCCs the sequences of the expressed ine mRNAs, which must bear host sequences between the HBV sequence and poly(A) tail. Using these cloned mRNA sequences, we will construct vectors bearing the entire large envelope protein coding sequence, and thus expressing all three HBV envelope proteins, large, middle and small (LMS vectors). Using these LMS vectors, we will determine for the first time whether HBV integrant-derived envelope proteins support the efficient assembly of infectious HDV, and thus ensure in vivo the completion of the HDV life cycle. This study will advance our understanding of the mechanism of the maintenance of chronic HDV infection and it may justify the use of HDV-targeting drugs (in addition to anti-HBV therapies) for chronic HBV/HDV carriers.